低温等离子体促进人牙龈上皮细胞在钛表面的黏附  

Promoting the adhesion of human gingival epithelial cells on titanium surface by non-thermal atmospheric plasma irradiation

作　　者：张敏[1] 敖小刚 郑铮 陈文川 Zhang Min;Ao Xiaogang;Zheng Zheng;Chen Wenchuan(State Key Laboratory of Oral Diseases&National Clinical Research Center for Oral Diseases&Dept.of Prosthodontics,West China Hospital of Stomatology,Sichuan University,Chengdu 610041,China)

机构地区：[1]口腔疾病研究国家重点实验室国家口腔疾病临床医学研究中心四川大学华西口腔医院修复科,成都610041

出　　处：《华西口腔医学杂志》2022年第3期285-292,共8页West China Journal of Stomatology

基　　金：四川大学华西口腔医院探索与研发项目(LCYJ2019-13);成都市科学技术局技术创新研发项目(2019-YD05-01328-SN)。

摘　　要：目的研究低温等离子体(NTAP)处理人牙龈上皮细胞(HGECs)后的生物学行为。方法将HGECs传代培养,待生长活性最佳的第3~5代细胞,消化重悬后经NTAP处理适当时间(0,10,20,30,60 s)后,接种在钛盘表面,加入口腔角质细胞培养基(OKM),1%双抗、5%CO_(2)和37℃孵箱条件下培养不同时间(4,12,24,48 h)贴壁生长,每组中各培养时间设置5个复孔。使用细胞计数试剂盒-8(CCK-8)评估各组黏附细胞的数量;扫描电子显微镜(SEM)观察各组细胞在钛片表面的形态;实时荧光定量聚合酶链式反应(qRT-PCR)检测各组细胞层粘连蛋白α3(Lamininα3)、整合素蛋白β4(Integrinβ4)和网蛋白(Plectin)黏附相关基因的表达情况以及Western blot观察各组细胞黏附相关蛋白表达的变化情况。结果在0~20 s内,随着NTAP作用时间的增加,细胞的黏附数量增加;但在20~60 s内,随着作用时间的增加,细胞的黏附数量逐渐减少:证明NTAP处理时间为20 s时最有利于HGECs在钛表面的黏附。CCK-8显示:在设定的培养时间下(20 s),随着培养时间延长,对照组和NTAP组细胞在钛表面的黏附数量增加。与对照组相比,在各培养时间点NTAP组细胞在钛表面的黏附数量更多(P<0.05)。SEM显示:NTAP组细胞在钛表面更明显地表现出不规则的多边形、突起与伪足较多及更大的细胞扩散面积。qRT-PCR显示:NTAP组细胞在钛表面的Lamininα3、Integrinβ4和Plectin黏附相关基因表达都高于对照组(P<0.05)。Western blot也证实:NTAP组细胞在钛表面上Lamininα3、Integrinβ4和Plectin黏附相关蛋白的表达均高于对照组。结论HGECs受NTAP处理后,结果表明20 s的作用时间能最大程度增加细胞在钛表面的黏附数量和改变黏附形态,同时能够显著上调Lamininα3、Integrinβ4和Plectin黏附相关基因和蛋白的表达,进而能促进HGECs对钛表面的生物封闭作用。Objective This work aimed to study the biological behavior of human gingival epithelial cells(HGECs)irradiated by non-thermal atmospheric plasma(NTAP)on a titanium surface.Methods Cultured HGECs(3⁃5 generations)with the best activity were digested and treated for varying times(0,10,20,30,and 60 s)by NTAP and then seeded on the surface of a titanium disc.The HGECs were cultured in oral keratinocyte medium and 1%penicillin/streptomycin solution.The cells were kept in an atmosphere of 5%CO_(2) at 37℃and incubated for different times(4,12,24,and 48 h;n=5).Cell counting kit-8(CCK-8)was used to detect cell adhesion capacity.Scanning electron microscopy(SEM)was conducted to observe the morphology of cells on titanium plates.Quantitative real-time polymerase chain reaction(qRT-PCR)and Western blot were used to evaluate the gene expression of adhesion-related molecules,such as Lamininα3,Integrinβ4,and Plectin.Results The number of adhered cells increased at 0⁃20 s,whereas that gradually decreased at 20⁃60 s.Therefore,cell culture at the two time points showed that HGECs adhesion reached the maximum when NATP was irradiated for 20 s.Compared with the control group,more cells in the treatment group adhered to the titanium surface at each time point(P<0.05).Cells in the treatment group showed more irregular polygons,more protrusions and pseudopods,and a larger cell diffusion area on the titanium surface than those in the control group.qRT-PCR showed that the expression levels of Lamininα3,Integrinβ4,and Plectin adhesion-related genes on the titanium surface in the treatment group were higher than those in the control group at each culture time point(P<0.05).Western blot showed that the expression levels of Lamininα3,Integrinβ4,and Plectin adhesion-related proteins on the titanium surface were higher in the treatment group than in the control group at 4 and 12 h.Conclusion After NTAP treatment,the results showed that 20 s of treatment time could maximize the number of adhered cells on the titanium surface;change

关 键 词：低温等离子体 人牙龈上皮细胞 黏附 钛表面 生物封闭 

分 类 号：R783.2[医药卫生—口腔医学]